There is mounting experimental support for a central role of mutations leading to constitutive activation of effectors signaling along the MAP kinase pathway in the pathogenesis of papillary thyroid carcinomas. Thus, there is practically no overlap between PTC with RET/PTC, NTRK, RAS or BRAF mutations, which altogether are found in >70% of cases. The lack of concordance for these mutations provides compelling genetic evidence for the requirement of this signaling system for transformation to PTC. This represents a unique paradigm of human tumorigenesis through mutation of multiple effectors lying in tandem. This is also of significance for development of novel therapies because if confirmed, selective kinase inhibitors acting distally in the pathway may prove to be efficacious for the majority of PTCs. Therefore, the core hypothesis of this proposal is that unregulated activation of MAP-kinase components is required for thyroid cell transformation to PTC, and that BRAF is a central node in the pathway. We will also test the hypothesis that continued BRAF activation is required for tumor maintenance. The following specific aims will be pursued: 1) Establish requirement of BRAF for RET/PTC-mediated transformation in vivo. 2) Determine whether continued expression of oncogenic BRAF is required for tumor maintenance. 3) Determine the role of the specific ERK phosphatases MKP-3 and DUSP5 in ERK activation and thyroid cell transformation after expression of RET or BRAF oncoproteins. 4) Determine the expression profile of thyroid PCCL3 cells after conditional expression of RET/PTC, BRAFV600E, or RET/PTC with BRAF-RNAi. The goal of this final aim is to identify common gene sets regulated similarly by RET/PTC and BRAF, as well as those that are distinct and may point to mechanisms accounting for phenotypic differences between PTCs associated with each of these oncoproteins.